The name of this project has been changed to reflect its evolution and a second related project has been discontinued as a separate entity and is now part of the work described here I study the transcriptional regulation of development in Drosophila. My approach is to look at the developmental effects of mutations in the transcriptional machinery itself, and in transcription factors and cell signaling molecules. Prior to arriving at NIH, I had isolated three extragenic mutations that enhance and 41 that suppress specific mutant alleles of RNA polymerase II subunits. These mutations define genes that encode proteins functionally interacting with RNA polymerase II in vivo, and might include: 1) general RNA polymerase II subunits, 2) developmentally regulated subunits, 3) proteins that modify polymerase activity, 4) proteins that regulate specific gene expression during development, and 5) proteins that maintain chromatin structure or nuclear architecture. I began analyzing the mutations since my arrival at NIH as a principal investigator in the beginning of 1992. In addition, I have initiated work on a key developmental transcription factor, dTcf, to determine how it signals RNA polymerase II. My research group has analyzed suppressor mutations of 4 different mutant alleles of RNA polymerase II. The interacting mutations identify specific domains in RNA polymerase II. For example, a mutation in the conserved F region of the largest subunit is suppressed by other mutations in F or by mutations in the second largest subunit in its conserved regions E-F. No other regions of polymerase subunits were identified. The original mutation is defective in elongation or termination and the suppressor mutations also affect this step in transcription. These results suggest that the identified domains are critical for elongation and/or termination. In addition, we have identified a new gene that interacts with these same mutations but does not map near a known subunit of RNA polymerase II. We are cloning this gene and suggest it may be an elongation or termination factor for RNA polymerase II.My research group has cloned another gene, prospero, which encodes an RNA polymerase II interacting protein. This gene was identified because an elongation or termination defective RNA polymerase II restored viability to a prospero mutant. Prospero encodes a homeodomain transcription factor required for the differentiation of glial cells in the Drosophila nervous system. Only one prospero allele, a 30 amino acid truncation of the conserved C-terminal Prospero domain, was suppressed. We have used expression studies to show this region has two functional domains. The N-terminus results in the nuclear export or cytoplasmic retention of a fusion protein. Addition of the C-terminal 30 amino acids allows the fusion protein to accumulate in the nucleus. We have also shown that the homeodomain can function as a transcriptional repressor.We have identified mutations in the Drosophila ortholog of the vertebrate transcription factors Tcf/Lef (T cell factor/lymphocte enhancer binding protein). Previously, Tcf and Lef had been identified by their differential expression and DNA binding during T cell and B cell differentiation. I collaborated in the cloning of dTcf in Drosophila to determine the first in vivo function of Tcf. Experiments using yeast two-hybrid experiments and ectopic expression in Xenopus suggested that Tcf/Lef family members interact with Armadillo/?-catenin to transduce Wingless/WNT signals during development. In addition, elevated levels of Tcf/?-catenin are observed in human colon cancer and melanoma. Using Drosophila genetics, we demonstrated that dTcf is the effector of wingless signaling in vivo. These results suggest that dTcf can be used to identify developmental targets and mis-expressed genes responsible for cancer.We demonstrated that both in vitro and in vivo dTcf interacts with Armadillo/?-catenin to transduce Wingless/WNT signaling. We are now focusing on the regulation and functioning of dTcf. We are generating antibodies to the Drosophila protein and inducing more mutations. We have also identified a direct target of dTCF regulation as decapentaplegic (dpp), an ortholog of transforming growth factor ?. We showed that two dTCF binding sites in dpp are required for repression of the gene in vivo. These results suggest that derepression by wingless/WNT signaling might be as important as activation in regulating development. My goal is to determine how Tcf signals RNA polymerase II in vivo, how it is regulated during development and to develop a Drosophila model for studying colon and other cancers.